Current estimates are that approximately 1% of all Mammalian genes code for integral plasma membrane proteins that belong to the superfamily of G protein-coupled receptors (GPCRs). To understand how these receptors function at a molecular level is the major focus of this research program. By using a combined molecular genetic/biochemical approach, the molecular mechanisms involved in GPCR folding and assembly, GPCR activation, and ligand/receptor/G protein interactions were explored. For these studies, different muscarinic acetylcholine and vasopressin receptor subtypes served as model systems. To elucidate the molecular basis underlying the selectivity of receptor/G protein interactions, we employed a novel experimental approach involving the coexpression of mutant GPCRs with hybrid G protein subunits. Using this strategy, we were able identify specific, functionally critical receptor/G protein contact sites, using the m2 and m3 muscarinic receptors as model systems. We also demonstrated that the receptor coupling selectivity of Galphas and Galphaq can be changed by single amino acid substitutions within the C-terminal portion of these proteins. Interestingly, the N-termini of Galphaq and Galpha11 differ from those of other Galpha subunits in that they display a unique, highly conserved six-amino acid extension. Coexpression studies with different GPCRs and wild type and mutant Galphaq subunits showed that this structural element is critical for constraining the receptor coupling selectivity of Galphaq/11, indicative of a novel mechanism by which the fidelity of receptor/G protein interactions can be regulated. The structural changes that accompany receptor activation are not well understood at present. Biophysical and biochemical studies with purified GPCR proteins should be of great importance to provide answers to these questions. We are currently in the process of developing a purification scheme for muscarinic receptors expressed in Sf9 insect cells, taking advantage of different epitope tags. In addition, we have created mutant muscarinic receptors in which (most of) the naturally occurring Cys residues were removed by site-directed mutagenesis. Unique Cys residues can now be substituted into defined positions of the "Cys-less" receptor proteins, followed by their modification with Cys-specific modifying agents carrying "environment-sensitive" reporter groups such as fluorescence markers or spin labels. This approach should lead to novel insights into GPCR structure and the dynamic processes that accompany GPCR activation.